Superior and Novel Detection of Small, Polar Metabolites in Saline Samples Using Pre-Extraction Benzoyl Chloride Derivatization and Ultra-High Performance Liquid Chromatography Tandem Mass Spectrometry (UHPLC MS/MS).

Dissolved metabolites support microbial systems via nutritional and energetic utilization and chemical signaling. However, the full scope and magnitude of these processes in marine systems is unknown largely because the mass spectrometric identification and quantification of these compounds from saline matrices has been hampered by poor extraction of small, polar compounds using common solid phase extraction resins (e.g. PPL resin, Bond Elut, Agilent). Here we utilized pre-extraction derivatization to improve the extraction, quantification, and chromatographic resolution of targeted dissolved metabolites in seawater and saline culture medium detected with ultra-high performance liquid chromatography electrospray ionization tandem mass spectrometry (UHPLC-ESI-MSMS) with Orbitrap detection. Metabolites were derivatized with benzoyl chloride by their primary and secondary amine and alcohol functionalities and quantified using stable isotope-labeled internal standards (SIL-IS) produced from 13C-labeled benzoyl chloride. We optimized derivatization, extraction, and sample preparation for field and culture samples, and we evaluated matrix-derived biases. This method quantifies 73 metabolites in seawater, of which 50 have an extraction efficiency <2% without derivatization. This method is sensitive (detection limits = pM to nM) and substantially improves sample throughput by shortening processing time ~10-20x